This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
An important component of cellular membrane, cholesterol controls physical properties of the membrane and contributes to specific membrane structures such as lipid rafts. Inside cells, cholesterol plays an important role in various signaling pathways and serves as the precursor for signaling molecules, and modifies specific proteins, such as hedgehog, to control protein trafficking and activity. The distribution of cholesterol in a living cell is highly regulated. Intracellular cholesterol is stored in lipid droplets (LDs) in the form of cholesteryl ester to avoid the toxicity caused by free cholesterol. Dysregulation of cholesterol metabolism and/or trafficking has been linked to various diseases, including atherosclerosis, Niemann-Pick type C (NP-C) disease, and various cancers.
Intracellular cholesterol transport and metabolism have been studied extensively using various reporter molecules, including cholesterol binding molecules and cholesterol analogs. Cholesterol binding molecules, such as cholesterol oxidase, filipin, and perfringolysin O derivatives, are commonly used to study steady-state distribution of cholesterol in fixed cells and tissues. Fluorescent cholesterol, including intrinsic fluorescent sterols (DHE for dehydroergosterol) and fluorophore-tagged analogs (NBD-cholesterol and BODIPY-cholesterol) are widely used in vitro and in vivo. Radiolabeled cholesterol or its precursors are used in biochemical studies of metabolism and trafficking of cholesterol.
These current cholesterol assays have limitations. Cholesterol oxidase is commonly used in fluorometric or colorimetric assays to quantify total cholesterol in homogenized cells. Radiolabeled cholesterol has to be used in combination with separation methods to determine intracellular cholesterol distribution indirectly. For imaging purpose, filipin is the most commonly used molecule for visualizing distribution of free cholesterol, but it is only applicable to fixed cells or tissues with moderate specificity because filipin also labels other lipids. Fluorescent BODIPY-cholesterol is known to cause perturbations due to bulkiness of the fluorophore. DHE has the closest structure as cholesterol, but its fluorescence undergoes rapid photo-bleaching, which impedes real-time observation of cholesterol trafficking There exists a need for new technologies that allow for real time imaging of cholesterol transport and low toxicity in live cells.